α-PD-1 therapy elevates Treg/Th balance and increases tumor cell pSmad3 that are both targeted by α-TGFβ antibody to promote durable rejection and immunity in squamous cell carcinomas

Abstract

Background Checkpoint blockade immunotherapy has improved metastatic cancer patient survival, but response rates remain low. There is an unmet need to identify mechanisms and tools to circumvent resistance. In human patients, responses to checkpoint blockade therapy correlate with tumor mutation load, and intrinsic resistance associates with pre-treatment signatures of epithelial mesenchymal transition (EMT), immunosuppression, macrophage chemotaxis and TGFβ signaling. Methods To facilitate studies on mechanisms of squamous cell carcinoma (SCC) evasion of checkpoint blockade immunotherapy, we sought to develop a novel panel of murine syngeneic SCC lines reflecting the heterogeneity of human cancer and its responses to immunotherapy. We characterized six Kras-driven cutaneous SCC lines with a range of mutation loads. Following implantation into syngeneic FVB mice, we examined multiple tumor responses to α-PD-1, α-TGFβ or combinatorial therapy, including tumor growth rate and regression, tumor immune cell composition, acquired tumor immunity, and the role of cytotoxic T cells and Tregs in immunotherapy responses. Results We show that α-PD-1 therapy is ineffective in establishing complete regression (CR) of tumors in all six SCC lines, but causes partial tumor growth inhibition of two lines with the highest mutations loads, CCK168 and CCK169. α-TGFβ monotherapy results in 20% CR and 10% CR of established CCK168 and CCK169 tumors respectively, together with acquisition of long-term anti-tumor immunity. α-PD-1 synergizes with α-TGFβ, increasing CR rates to 60% (CCK168) and 20% (CCK169). α-PD-1 therapy enhances CD4 + Treg/CD4 + Th ratios and increases tumor cell pSmad3 expression in CCK168 SCCs, whereas α-TGFβ antibody administration attenuates these effects. We show that α-TGFβ acts in part through suppressing immunosuppressive Tregs induced by α-PD-1, that limit the anti-tumor activity of α-PD-1 monotherapy. Additionally, in vitro and in vivo, α-TGFβ acts directly on the tumor cell to attenuate EMT, to activate a program of gene expression that stimulates immuno-surveillance, including up regulation of genes encoding the tumor cell antigen presentation machinery. Conclusions We show that α-PD-1 not only initiates a tumor rejection program, but can induce a competing TGFβ-driven immuno-suppressive program. We identify new opportunities for α-PD-1/α-TGFβ combinatorial treatment of SCCs especially those with a high mutation load, high CD4+ T cell content and pSmad3 signaling. Our data form the basis for clinical trial of α-TGFβ/α-PD-1 combination therapy (NCT02947165). Electronic supplementary material The online version of this article (10.1186/s40425-018-0493-9) contains supplementary material, which is available to authorized users.

Integrin αvβ8 on T cells is responsible for suppression of anti-tumor immunity in multiple syngeneic models and is a promising target for tumor immunotherapy

“…The surviving mice that received either monotherapy or combination therapy were re-challenged with the same tumor cells at >50 days after initial therapy in both the CCK168 and CT26 models. There was minimal tumor growth observed, less than 100 mm 3 , and all tumors that did grow out subsequently regressed despite no further treatment, suggesting that successful treatment with ADWA-11 can lead to long-term anti-tumor immunity, as has been previously described for other immunomodulators, including TGFβ blockade ( Figure 3C) (Dodagatta-Marri et al, 2019) .…”

“…Effects of combinatorial therapy of EMT6 mammary and TRAMPC2 prostate carcinoma models We began by examining the effects of ADWA-11, alone, or in combination with anti-PD-1, in established syngeneic tumor models of squamous cell carcinoma (CCK168 cells) and mammary carcinoma (EMT6 cells) ( Figure 1A), two models in which systemic blockade of TGFb has been shown to enhance responses to immunotherapy (Dodagatta-Marri et al, 2019;Mariathasan et al, 2018). We injected CCK168 cells subcutaneously into the flanks of syngeneic mice or EMT-6 cells directly into the fourth mammary fat pad, and allowed tumors to grow to 65mm 3 before beginning antibody therapy.…”

“…Previous studies have shown that inhibition of TGFb signaling can enhance responses to checkpoint inhibitors (Dodagatta-Marri et al, 2019;Mariathasan et al, 2018;Tauriello et al, 2018). However, TGFβ plays important homeostatic roles in many biological systems, so effective systemic targeting of TGFβ signaling would likely present challenges due to unwanted side effects (Akhurst and Hata, 2012;Flavell et al, 2010;Yang et al, 2020).…”

Lessons learned from SMAD4 loss in squamous cell carcinomas

“…Because single therapeutic approaches are often met with tumor resistance, it is more likely that a combination of therapies targeting multiple SMAD4 deficient‐associated mechanisms in addition to standard care approaches, such as olaparib with PD‐1/PD‐L1 blockade and radiation, would be more effective at reducing SMAD4 deficient SCC tumor burden. Alternatively, a combinatory approach with PD‐1/PD‐L1 blockade and a TGFß inhibitor is compelling as PD‐1/PD‐L1 blockade can induce competing TGFß‐driven immune suppression …”

“…Alternatively, a combinatory approach with PD-1/PD-L1 blockade and a TGFß inhibitor is compelling as PD-1/PD-L1 blockade can induce competing TGFß-driven immune suppression. 73…”